Rotavirus (RV) causes severe diarrhea in children with significant morbidity and mortality. While the current live oral RV vaccines are highly effective in developed nations, their efficacies are impaired in developing countries, where most RV morbidity and mortality remain and thus where RV vaccines are needed the most. Factors leading to the impaired efficacies include microbiota dysbiosis, concurrent uses of poliovirus and other oral vaccines, enterovirus infections, and malnutrition that impact intestinal conditions and thus the efficacies of the oral RV vaccines. As a result, parenteral RV vaccine approaches are called to improve the efficacy in the developing countries. To this end, we have developed an innovative, nanoparticle-based S60-VP8* RV vaccine that would meet such calls. The recombinant, nonreplicating nature of our vaccine and its parenteral delivery method will also reduce vaccine production cost and prevent intussusception risk of the live RV vaccines for better safety and cost-effectiveness. The bioengineered S60-VP8* nanoparticle is self-assembled, easily produced, highly stable, and extremely immunogenic, and therefore, an excellent RV vaccine candidate. Each S60-VP8* nanoparticle contains a 60-valent norovirus inner shell and 60 surface-displayed RV VP8* antigens. The viral receptor-binding VP8* is an ideal RV vaccine target, because antibodies elicited by nature RV infections are mostly VP8*-specific and vast majority of VP8*-directed antibodies neutralized RV infections. As a proof of concept, we have shown the high immunogenicity, neutralization, and protection of the S60-VP8* nanoparticle displaying the predominant P[8] RV VP8* in mouse model. In this application we will produce a cocktail S60-VP8* nanoparticle vaccine displaying RV VP8*s of the globally predominant P[8], P[4], P[6], and P[11] RVs and define its safety, immunogenicity, and protective efficacy using the mouse and the highly relevant gnotobiotic (Gn) pig models. This is the first nanoparticle-based cocktail RV vaccine covering all four predominant P type RVs and therefore will provide a broad protection against RV infections in both developed and developing nations. The outcomes from both small and large animal models will prove the usefulness and thus facilitate future clinical trials of our S60-VP8* nanoparticle vaccine. Two major lines of experiments will be performed in this application. First, we will produce the cocktail S60-VP8* nanoparticle vaccine covering the four predominant RV P types (P[8]/P[4]/P[6]/P[11]) and evaluate the cross-P type immune responses, neutralizations and protections, as well as delineate the immune mechanisms of the cocktail vaccine in mice. Second, we will determine the safety, cross-P type immunogenicity, and broad neutralization/protection of the cocktail nanoparticle vaccine compared with the currently implemented live RV vaccines in the Gn pig human RV challenge model. Mechanistic study will also be performed to understand the broad immune response and neutralization/protection of the cocktail vaccine in Gn pigs. Given our strong preliminary data and the long collaboration history of the research team with outstanding productivity track records, we will fulfill the goals of this project.